A particularly important element of addiction is the potential for influencing gene expression by environment and experience. The development of drug dependency and addiction is a function of both genetic predisposition and environmental factors. Thus, posttranscriptional mechanisms provide particularly potent machinery for addictive processes. We have recently discovered an epigenetic mechanism involving a microRNA (miR-9) in the adaptive changes in a neuronal ion channel (BK), in response to alcohol exposure. BK is a Ca- and voltage-gated potassium channel very important in neuronal excitability, and is a well-described target of alcohol action and tolerance. The miRNAs are small (19-25nt) non-coding RNAs which represent a class of endogenous post-transcriptional repressors of gene expression. A rise in miR-9 elicited by alcohol results in the selective degradation of alcohol-sensitive splice variants of the BK channel, causing a changed landscape in which the majority of the remaining BK channel isoforms are alcohol-resistant. In the proposed research, we will use molecular biology techniques to examine the mechanisms by which alcohol upregulates miR-9, and the characteristics of that upregulation. These studies will be augmented with a combination of electrophysiological and molecular imaging approaches to examine the consequences of miR-9 upregulation on BK channel mRNA alternative splicing, and on the ultimate disposition and function of the BK channel protein after alcohol exposure . Particular hypotheses to be explored include: in Aim 1: The rapid upregulation and persistence of miR-9 is a consequence of the increase in miR-9 production from its immediate precursor (pre-miR-9), and is controlled by the alcohol exposure protocol; in Aim 2: Manipulation of miR-9 will result in changes in BK mRNA consistent with a causative role; A demonstrable relationship will be evident between alcohol induced changes in miR-9, and BK mRNA amount and splicing; in Aim 3: Manipulation of miR-9 will result in changes in BK channel electrophysiology and morphology consistent with a causative role; A demonstrable relationship will be evident between alcohol induced changes in miR-9 and mRNA after alcohol exposure, and subsequent changes in BK channel proteins monitored by electrophysiology and molecular imaging. PUBLIC HEALTH RELEVANCE The presence of enhanced acute behavioral tolerance in humans can serve as a marker for likelihood of the development of alcoholism. One challenge to our understanding of alcohol abuse and addiction is to identify the adaptations within individual molecules that underlie tolerance. In this project we will explore a novel mechanism, acting on gene products, which will provide important insights in our quest for this understanding.